Antibody Responses Against Different Pathogenic Fungi

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2.1. Cryptococcus neoformans

C. neoformans is a ubiquitous environmental yeast that is unique among the pathogenic fungi because it has a polysaccharide capsule. Serological studies suggest that C. neoformans infections are very common among immunocompetent individuals, including children, but disease is rare (Goldman et al., 2001). However, as an opportunistic pathogen C. neoformans is able to cause life-threatening infections, mainly in patients with cellular immune deficiencies, most notably AIDS (Perfect and Casadevall, 2002). Current therapy for cryptococcosis is sub-optimal and human infection is often incurable (Perfect and Casadevall, 2002). Since the majority of patients with cryptococcal infection are immunosuppressed a logical approach to the improvement of treatment outcomes is through enhancing host immune responses. Host defense against C. neoformans is believed to depend primarily on the cellular response (Levitz, 1992). Early experiments with polyclonal sera produced conflicting evidence for and against the importance of antibody immunity in host defense during cryptococcosis. However, numerous studies have now convincingly demonstrated that antibody-mediated immunity may play a role in resistance against cryptococcal infection (Casadevall, 1995).

C. neoformans cells are surrounded by a polysaccharide capsule that is antiphago-cytic and represents a major virulence factor (McFadden and Casadevall, 2001; Perfect et al., 1998). During human infection, the capsular polysaccharide, glucuronoxylomannan (GXM), accumulates in tissue, which negatively impacts host immunity (Murphy, 1999; Vecchiarelli, 2000). The majority of patients with C. neoformans infections have a high concentration of serum polysaccharide antigen but low titers of serum antibodies to the capsular polysaccharide. Experiments with monoclonal antibodies to GXM have demonstrated the existence of protective, non-protective and even deleterious antibodies, thus providing an explanation for the earlier divergent results using polyclonal sera (Casadevall, 1995). Several groups have now convincingly demonstrated that administration of antibodies to GXM can modify the course of infection to the benefit of the host (Dromer et al., 1987; Fleuridor et al., 1998; Mukherjee et al., 1992; Sanford et al., 1990). Antibodies have been shown to enhance opsonization, activate the complement system, increase phagocytosis (thereby increasing the number of organisms killed) and also promote the clearance of polysaccharide antigen from the serum. Overall, in animal models administration of anti-GXM antibodies prolonged survival, reduced tissue fungal burden, promoted granuloma formation, and enhanced the killing of C. neoformans by antifungal agents. Efficacy of mAb-treatment against C. neoformans in mice is dependent on isotype, specificity (which in turn is affected by immunoglobulin variable region usage, somatic mutation and constant region usage), binding characteristics, dose and infecting inoculum, timing of administration, fungal strain, mouse strain and availability of T cells and other mediators of cellular immunity (reviewed in (Casadevall and Pirofski, 2005)). Altogether, the data generated indicates that anti- GXM antibodies mediate protection by immunomodulatory effects, thereby linking antibody efficacy to the overall host immune response (Casadevall et al., 2002; Feldmesser et al., 2002). Based on these observations, MAb 18B7, a murine IgG1 developed by the Casadevall group, is currently in clinical trials which represents the first application of mAb therapy for the treatment of a fungal infections in humans (Casadevall et al., 1998; Larsen et al., 2005).

In addition, the protective efficacy of anti-GXM provides a rational basis for the development of vaccines against C. neoformans infections if such a vaccine could induce similar antibodies (Casadevall et al., 2002). A GXM-tetanus toxoid (GXM-TT) conjugate vaccine protected mice against infection (Devi, 1996). Also, peptide mimetics of GXM oligosaccharide epitopes have been identified using random peptide phage display libraries (Fleuridor et al., 2001; Valadon et al., 1998). A peptide mimetic of a human IgM proved to be a GXM mimotope capable of eliciting an antibody response to GXM (Fleuridor et al., 2001). Importantly, vaccination with this mimotope prolonged survival of mice infected with C. neoformans.

Melanin synthesis has also been associated with virulence for this fungus (Casadevall et al., 2000). C. neoformans cells synthesize melanin during infection and this pigment protects the fungus against immune defense mechanisms. Passive immunization with mAbs to melanin prolonged the survival of and reduced the fungal burden in infected mice (Rosas et al., 2001). The anti-melanin antibodies reduced the growth rate of in vitro-melanized C. neoformans cells, suggesting a new mechanism of antibody-mediated protection. Similarly, antibodies to cell wall glucosylceramide exhibit fungistatic effects (Rodrigues et al., 2000). Antibody responses to several cytosolic and membrane proteins have been associated with increased survival in murine models (Neuville et al., 2000). Interestingly, the antibody response to protein antigens was bimodal, with nonsurvivors mounting a strong antibody response early during the acute phase, but a majority of survivors producing antibodies later, during the chronic phase of the infection. These observations highlight the existence of additional targets for the humoral response during cryptococcosis.

2.2. Candida albicans

Candida albicans is the most common fungal pathogen of humans. Although in normal individuals this microorganism is a commensal of mucosal surfaces, in patients with predisposing conditions it is able to cause a variety of infections that range from superficial (oral and vaginal) candidiasis to life threatening disseminated candidiasis. Although C. albicans remains the most frequent causative agent of candidiasis, other species have been increasingly associated with infections in an expanding population of immunocompromised patients. Morbidity and mortality rates associated with candidiasis remain unacceptably high, the main reasons being the difficulties encountered in the diagnosis and treatment of this type of infections (Banerjee et al., 1991; Pfaller et al., 1999; Pfaller et al., 1998; Viudes et al., 2002).

As a result of its commensal status anti-Candida antibodies have been shown to be ubiquitous in human sera, presumably because the immune system can be stimulated as a result of colonization by C. albicans in the absence of disease (Domer, 1989; Lopez-Ribot et al., 2004; Martinez et al., 1998; Reiss and Morrison, 1993), starting early during infancy. During the host-fungus relationship, both as a commensal and during infection, C. albicans antigens elicit strong immune responses, including production of antibodies. Cytosolic components, cell wall and other secreted moieties, both protein and carbohydrate in nature, represent the major immunogenic components during candidiasis as manifested by proteomic analyses of antibodies present in sera from patients and animal models (Lopez-Ribot et al., 2004; Martinez et al., 1998; Pitarch et al., 2004; Pitarch et al., 2001; Pitarch et al., 1999). Despite the complex antigenic make-up and considerable heterogeneity of the antibody responses to candidal antigens in humans (Chaffin et al., 1998; Lopez-Ribot et al., 2004; Martinez et al., 1998), several immunodominant antigens have been identified, including glycolytic enzymes (enolase), heat shock proteins, mannans and manno-proteins. The role of antibody immunity in protection against candidiasis has been controversial, the antibody response against Candida is complex, with the presence in immune sera of protective, non-protective, and deleterious (infection-enhancing) antibodies (Casadevall, 1995; Fernandez-Arenas et al., 2004a; Fernandez-Arenas et al., 2004b; Lopez-Ribot et al., 2004; Martinez et al., 1998). Fernandez-Arenas et al described two categories of serum, protective and non protective, and the antibody profiles were analyzed by a proteomic approach (Fernandez-Arenas et al., 2004a). Immunoglobulin levels (particularly IgG2a) in the protective sera were significantly higher when compared with the non protective sera. The pattern of a "non protective" profile was composed of enolase (Eno1p), transketolase (Tkl1p), heat shock protein (Hs78p) and methionine synthase (Met6p), only antibodies against enolase were IgG2a isotype. The pattern of a "protective" sera, on the other hand, was composed of antibodies against the following antigens: several isoforms of enolase (Eno1p), pyruvate decarboxylase (Pdc11p), pyruvate kynase (Cdc19p), a protein of the 40S ribosomal subunit (Bel1p), triosephosphate isomerase (Tpi1p), DL-glycerol phosphatase (Rhr2p) and fructose-bisphosphate aldolase (Fba1p), and all these antibodies are IgG2a isotype.

As a result, early immunoprotection experiments using polyclonal antibodies often lead to inconclusive observations (Casadevall, 1995). However, recent evidence clearly demonstrates that antibodies with defined specificities show different degrees of protection against systemic and mucosal candidiasis (De Bernardis et al., 1997; Han and Cutler, 1995; Matthews and Burnie, 2001), favoring the host during the course of infection (Casadevall, 1995; Lopez-Ribot et al., 2004; Martinez et al., 1998). The exact mechanisms by which these antibodies protect against Candida infection are unknown but are likely to include the inhibition of adhesion or germ tube formation, opsonization, neutralization of virulence-related enzymes, and direct candidacidal activity (Casadevall, 1995; Martinez et al., 1998). Further data supporting the protective role of antibodies during candidiasis are: i) the fact that functional B cells are required to protect mice from a primary intravenous challenge with C. albicans (Wagner et al., 1996) and ii) the fact that opsonizing antibodies may condition the nature of the dendritic cell interaction with fungi and the generation of memory antifungal immunity (Montagnoli et al., 2003). A recent work has addressed the importance of the collaboration between cellular and humoral responses, in the eliciting a long lasting and effective protection (Fernandez-Arenas et al., 2004b). The authors vaccinated mice with a series of "avirulent" mutants and using a proteomic approach were able to define an antibody pattern in the sera from effectively vaccinated animals surviving the infection that differed from the antibody pattern in non-protected mice. The use of this proteomic approach led to the identification of antigens eliciting protective IgG2a antibodies in vaccinated animals, which may represent excellent candidates for a future fungal vaccine (Fernandez-Arenas et al., 2004b).

Mannan is found in the cell wall as large N-linked and shorter O-linked mannooligosaccharides associated with mannoproteins. Anti-mannan antibodies are prevalent in human sera, including patients and normal population (Domer, 1989; Lopez-Ribot et al., 2004; Martinez et al., 1998; Reiss and Morrison, 1993). The antigenic specificity of serotypes A and B of C. albicans is determined by structural peculiarities of the carbohydrate moiety of mannans (Hasenclever and Mitchell, 1964). The mannan component is also involved in adhesive interactions (Chaffin et al., 1998). Han and Cutler immunized mice with a mannan fraction (previously encapsulated into liposomes) to induce protective antibody responses. In the same study, these authors tested two monoclonal antibodies specific for different mannan epitopes in the adhesin fraction. Both antibodies agglutinated Candida cells but only one of them protected mice against disseminated candidiasis. The protective antibody recognized the acid-labile adhesin while the non-protective antibody recognized a different epitope in the fraction (Han and Cutler, 1995; Han et al., 2000). It seems that the ability of the antibody to rapidly deposit high amounts of complement factor C3 onto the yeast cell wall is needed for protection against disseminated candidiasis (Han et al., 2001). Anti-mannan antibodies can also mediate protection in animal models of Candida vaginitis (De Bernardis et al., 2002; De Bernardis et al., 1997; Han et al., 1998). Combined detection of mannanemia and anti-mannan antibodies may have diagnostic value and also help monitor patients with candidiasis (Sendid et al., 2002).

Although P-glucans are present in greater abundance than mannan in the wall of C. albicans, they are immunologically less active (Chaffin et al., 1998; Martinez et al., 1998). However, it has been recently reported that anti-glucan antibodies contributed to the passive protection against Candida infection in an animal model (Bromuro et al., 2002). Also, P-glucan has recently been identified as a the receptor for the killer toxin produced by the yeast P. anomala (Guyard et al., 2002; Magliani et al., 2005). Interestingly protective yeast killer toxin-like antibodies ("antibio-bodies", KT-Abs), are able to exert a direct microbicidal activity by mimicking a killer toxin (PaKT) and its interaction with cell wall receptors on susceptible C. albicans cells. These antibodies are produced during the course of experimental and natural infections and can also be produced by idiotypic vaccination with a KT-neutralizing mAb, and they exert strong therapeutic activity against both mucosal and systemic candidiasis (Beninati et al., 2000; Magliani et al., 2005; Magliani et al., 2002; Magliani et al., 2004; Polonelli et al., 2000; Polonelli et al., 1996; Polonelli et al., 1994; Polonelli et al., 2003; Polonelli et al., 1997), and also different fungal infections, including aspergillosis (Cenci et al., 2002) and experimental models of Pneumocystis carinii pneumonia (Magliani et al., 2001).

Immunoblotting experiments with sera from patients suffering from systemic candidiasis showed the presence of a 47-kDa immunodominant antigen present in whole cell extracts of the fungus later identified as a heat-stable breakdown product of hsp90 with a cell wall location (Matthews and Burnie, 1989; Matthews et al., 1988b; Matthews et al., 1991b; Matthews et al., 1984). Antibodies to the 47-kDa antigen are present in serum samples from a high proportion of patients with chronic mucocutaneous candidiasis and AIDS (Matthews and Burnie, 1988; Matthews et al., 1988a; Matthews et al., 1991b). Patients who recover from invasive candidiasis generate a major antibody response to the 47-kDa component, whereas fatal cases seem to have little antibody or declining titers (Matthews and Burnie, 1988; Matthews et al., 1988a; Matthews et al., 1991b). In a mouse model of disseminated candidiasis, passive administration of sera from two infected patients containing anti-hsp90 antibodies led to decreased mortality. Epitope mapping of C. albicans hsp90 with patients' sera revealed that patients recovering from systemic candidiasis produce antibodies against both fungal-specific and conserved epitopes of hsp90 (Matthews et al., 1991a). A highly conserved epitope LKVIRK was recognized by sera from all patients with antibody to the 47-kDa antigen. When administered prophylactically, a monoclonal antibody raised against this epitope reduced mortality in a mouse model of hematogenously disseminated candidiasis (Matthews et al., 1995). Mycograb (NeuTec Pharma plc.) is a human genetically recombinant antibody against fungal hsp 90. Mycograb acts synergistically with amphotericin B and caspofungin both in vitro and in vivo. Based on these properties, Mycograb is now being assessed in preclinical trials in patients with invasive candidiasis (Matthews et al., 2003). In addition to hsp90, several members of the hsp70 family of proteins have been reported in the cell wall of C. albicans. It was reported that serum samples from both healthy people and patients suffering from candidiasis contained antibodies against the C-terminal portion of a member of the hsp70 family of proteins (Lopez-Ribot et al., 1996a). A more recent report demonstrated the elevated immunogenicity of hsp70, however, no protection against but rather some enhancement of Candida infection seemed to occur in a murine model of candidiasis after vaccination with recombinant protein (Bromuro et al., 1998). Immunization with a stress mannoprotein of >200 kDa from the cell wall of C. albicans, a major target of secretory IgA, led to the production of mAbC7. This mAb displays three different biological activities (inhibition of adherence, inhibition of germination and direct candidacidal activity) (Moragues et al., 2003), and also has tumoricidal activity (Omaetxebarria et al., 2005).

Perhaps the glycolytic enzyme enolase is the most immunodominant component in C. albicans and antibodies against enolase and other cytosolic and cell wall-associated glycolytic enzymes, including phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, fructose-bisphosphate aldolase, triose phosphate isomerase, phosphoglycerate mutase, pyruvate kinase, pyruvate decarboxylase and alcohol dehydrogenase, seem to be prevalent in sera from patients and animal models (Alloush et al., 1997; Chaffin et al., 1998; Lopez-Ribot et al., 2004; Martinez et al., 1998; Pitarch et al., 2004; Pitarch et al., 2001; Pitarch et al., 1999). Strockbine et al. (Strockbine et al., 1984) reported that sera from patients with disseminated candidiasis had circulating antibodies directed against a 48-kDa protein antigen, which was subsequently identified as enolase (Franklyn et al., 1990; Mason et al., 1989). A protective role for anti-enolase antibodies has been suggested since repeated administration of immune anti-enolase serum conferred partial protection against murine candidiasis (van Deventer et al., 1996). In a recent report mice immunized with enolase plus IL-12 showed increased antibody titres against enolase, as well as increased median survival time and decreased fungal burden in kidneys, although antibodies did not seem to play a role in protection (Montagnoli et al., 2004).

C. albicans secreted aspartyl proteinases (Saps) represent key virulence determinants during candidiasis. Saps are immunogenic and elicit mucosal and systemic antibody responses (Naglik et al., 2003). Perhaps the most promising results were reported by De Bernardis et al. (De Bernardis et al., 2002; De Bernardis et al., 1997), who showed that immunization with Sap2 antigen, or administration of an anti-Sap2 monoclonal antibody or anti-Sap2 antibody-containing vaginal fluids, partially protected rats against candidal vaginitis in an experimental model. One study analyzed the B-cell epitopes of Candida Sap proteins, particularly Sap2, and provided some information regarding the Sap2 epitopes recognized by serum IgG and IgM antibodies (Ghadjari et al., 1997).

The 58-kilodalton fibrinogen binding cell wall mannoprotein (mp58/Pra1p) belongs to a family of immunodominant fungal antigens and is expressed by fungal cells during infection (Casanova et al., 1992; Lopez-Ribot et al., 1996b). Immunoblotting analyses demonstrated the presence of antibodies against the mp58 in sera from patients with different types of candidiasis (Navarro et al., 1993). Continuous B-cell epitopes on the protein moiety of C. albicans mp58 species, have been recently identified in epitope mapping experiments using a complete set of overlapping dodecapeptides synthesized from amino acid sequence of the protein portion of mp58 as deduced from the DNA sequence of its encoding gene (FBP1/PRA1) (Viudes et al., 2004; Viudes et al., 2001). These experiments used sera from patients with candidiasis and from animal models and revealed the presence of multiple IgG-reactive continuous epitopes on the protein, expanding both the amino- and carboxy-terminal domains and several internal regions. A synthetic peptide corresponding to the last 10 amino acid residues at the C terminus of the protein elicited a strong antibody response in mice (Viudes et al., 2001). Patients who survived the infection displayed increased antibody reactivity towards the C-terminal epitope as compared to those succumbing to candidiasis (Viudes et al., 2004). Moreover, a monoclonal antibody directed towards this epitopic region conferred protection in serum therapy experiments in a murine model of hematoge-nously disseminated candidiasis (Viudes et al., 2004).

Other interesting use of antibodies as tools in the study of pathogenesis of candidiasis (and other fungal infections) is their use to screen expression libraries leading to the identification of genes encoding immunoreactive proteins and also those expressed in vivo during infection (Alloush et al., 1996; Nguyen et al., 2004). Also, the fact that a mAb recognizing a pH sensitive carbohydrate epitope on the surface of C. albicans cells selectively identifies invasive forms of candidiasis offers new hope for the development of effective diagnostic techniques that could discriminate between commensal status and active infection (Monteagudo et al., 2004).

2.3. Pneumocytis carinii

P. carinni is an opportunistic fungus that casues a potentially severe and fatal pneumonia (PCP) in a variety of patients with compromised immune status due to AIDS, chemotherapeutic regimes for cancer, immunosuppressive therapy for organ transplant and congenital immune diseases (Wazir and Ansari, 2004).

Roth and Janitschke demonstrated the formation of antibodies in mice, rats and rabbits immunized with different P. carinii antigens (Roth and Janitschke, 1991). Hyperimmune sera was highly effective at reducing the number of fungal organisms in early, intermediate, and advanced stages of PCP and was capable of increasing the mean life expectancy of infected mice (Roths and Sidman, 1993) and passive immunization with polyclonal antibodies in sera from animals that were allowed to recover from severe P. carinii pneumonia conferred protection to naive mice (Bartlett et al., 1998).

Gigliotti et al., developed a battery of monoclonal antibodies against P. carinii surface components (Gigliotti et al., 1986). Only one of these antibodies, an IgM, recognized P. carinii obtained from rabbits, ferrets, and human. Interestingly, passive immunoprophylaxis with this specific mAb conferred partial protection against PCP in animal models (Gigliotti and Hughes, 1988). This mAb recognizes a conserved epitope on a major mannosylated surface glycoprotein (gpA) of P. carinii (Haidaris et al., 1992). However, immunization with gpA produced an specific antibody response but did not protect mice from the development of PCP (Gigliotti et al., 1998). Subsequently this same group generated a panel of mAbs against P. carinii antigens other than gpA. Some of these mAbs were protective when administered intranasally, with two IgM antibodies recognizing and epitope on the kexin-like molecule (KEX1) and also the A12 antigen shared by mouse and human organisms leading to a reduction in fungal burden of more than 99% (Gigliotti et al., 2002). Epitope mapping with one of these mAbs indicated recognition of multiple proline-rich epitopes which may explain its degenerate recognition of multiple antigens on the surface of the fungus, including KEX1 and also the A12 antigen (Wells et al., 2004).

2.4. Aspergillus fumigatus

Over the past two decades Aspergillus fumigatus has become the most prevalent airborne fungal pathogen, causing severe and usually fatal invasive infections in immunosupressed patients (Denning , 1998; Marr et al., 2002; Patel and Paya, 1997). Invasive aspergillosis is now a major cause of death at leukemia treatment centers, and bone marrow and solid organ transplantation units (Denning , 1998; Marr et al., 2002; Patel and Paya, 1997). Other spp. of Aspergillus, such as A. terreus, A. flavus and A. niger can also cause invasive aspergillosis (Marr et al., 2002). This severe opportunistic fungal infection is characterized by a high mortality rate in these at-risk patients (Lin et al., 2001) (the crude mortality rate of invasive aspergillosis approaches 100%). Although rarely, in immunocompetent patients this fungus can also cause aspergilloma (an overgrowth of the fungus on the surface of preexisting cavities in the lungs of patients treated for tuberculosis) and allergic brochopulmonary aspergillosis (ABPA, a clinical condition observed among individuals exposed repeatedly to conidia and those suffering from atopic asthma or cystic fibrosis).

Most studies to date have examined antibodies against Aspergillus as contributing to allergic disease or because of their use in serodiagnostic techniques. Sera from most healthy individuals contain anti-Aspergillus antibodies due to continuous environmental exposure. In contrast to immunocompetent hosts, growth of A. fumigatus in the tissues of an immunocompromised host, who either lack a sufficient antibody response or who mount variable antibody response, is not correlated with an increase in anti-Aspergillus antibody titers. Indeed, presence of antibodies against Aspergillus in immunosuppressed individuals is more likely to represent circulating antibodies prior to the onset of immunosuppressive therapy rather than antibodies formed during invasive infection (Latge, 1999). Increasing antibody titers at the end of immunosuppression are normally indicative of recovery from invasive aspergillosis, whereas declining antibody levels are normally associated with poor prognosis. On the other hand, high antibody titers (precipitins) are normally detected in patients with aspergillomas, and those with ABPA show elevated levels of serum IgE (Jaques et al., 1995). Immunoblot analyses have been used to examine the antibody response during infection and to identify antigenic components of Aspergillus spp. These have also served as the basis for the development of serodiagnostic assays for the diagnosis of aspergillosis in immunocompetent hosts, although studies were complicated by the variability of antigenic extracts used. Important antigenic components identified include 58-kDa, 88-kDa and 18-kDa proteins, catalase, alkaline protease, a serine protease, a superoxide dismutase, elastase and catalase, among others (reviewed in (Latge, 1999)). More recently Denikus et al used antibodies in sera from infected rabbits to screen an A. fumigatus expression library, and identified thirty six antigens, most of which were associated with the cell surface, and included Asp f 16, hsp90 and enolase (Denikus et al., 2005). Importantly, the authors suggested that antibody production may be associated with the development of an early immune response and primary protection.

But without any question, a polysaccharide antigen, galactomannan (GM), is the most important antigen in Aspergillus. GM is a component of the Aspergillus cell wall, was the first antigen detected in experimentally infected animals and in patients with invasive aspergillosis (Latge, 1999). Recently, Stynen et al. have introduced a sandwich enzyme-linked immunosorbent assay (ELISA) that is based on the use of the rat monoclonal antibody EB-A2, which recognizes the (1 ^ 5)-8-D-galactofuranoside side chain of the GM molecule. Since each GM molecule harbors several epitopes, the same monoclonal antibody can function as capture and detector antibody (Stynen et al., 1995). The test is commercialized as Platelia Aspergillus EIA (Bio Rad), for the early diagnosis of invasive aspergillosis, which is of paramount importance since antifungal therapy must be begun promptly in these highly immunosuppressed patients to maximize success. Of note, this test could also be important for monitoring therapeutic responses (Bennett et al., 2003).

Presently very little is known about the potential of anti-Aspergillus antibodies to protect during infection. Although many mAbs against different Aspergillus antigens have been generated, the majority have not been tested for protective efficacy (Casadevall et al., 2002). Most attempts at passive transfer of protection with immune sera have been unsuccessful, although most studies did not examine different parameters known to affect outcome in other experimental systems. For example, one study used mAbs to elastase, which did not confer protection to immunosuppressed mice (Frosco et al., 1994). However, the possibility exists that antibodies to Aspergillus may mediate protection, for example, by modulating cellular immune responses, interfering with spore germination, increasing phagocytosis or neutralizing hydrolytic enzymes (Casadevall et al., 2002). For example, recent results suggest that the availability of opsonizing antibodies may condition the nature of the dendritic cell interaction with fungi, and play an important role in the generation of memory antifungal immunity against aspergillosis (Montagnoli et al., 2003).

2.5. Histoplasma capsulatum

H. capsulatum is a dimorphic fungus and is the most prevalent cause of fungal respiratory disease, infecting approximately 500,000 individuals in the US each year (Cano and Hajjeh, 2001). Infection usually results in a mild, often asymptomatic respiratory illness, but may progress to life-threatening systemic disease, especially in individuals with AIDS (Graybill, 1988). The clinical manifestations are principally caused by intracellular yeast forms that parasitize mammalian phagocytes.

The antibody response to natural infection by H. capsulatum in humans has been investigated (for a review see (Nosanchuk, 2005)), and is characterized by induction and increase in IgM in the first two weeks, followed by increasing titers of IgG and IgA antibodies (Chandler et al., 1969). The IgG fraction contains both complement-fixing and precipitating antibodies (Chandler et al., 1969). Experiments in animal models (mouse) indicate that antibody levels peak by day 21 (Fojtasek et al., 1993). Antibody detection may give a clue to the diagnosis of this infection, even in immunosuppressed hosts. Antibodies to the H antigen of histoplasmin develop during active histoplasmosis while antibodies to M antigen are indicative of prior infection and is the first to rise with seroconversion (Wheat et al., 1983). Nevertheless, limitations of serological detection of histoplasmosis include lack of humoral response in those patients with more severe immunosuppression and due to the lack of sensitivity or specificity of that response (Wheat et al., 1983). More useful, particularly in immunosuppressed hosts, is the use of antibodies for the detection of Histoplasma polysaccharide, which is detectable during disseminated infection and correlates with response to therapy (Wheat, 2003). This assay system developed by Wheat and colleagues, utilizes a polyclonal antibody for both the capture and detection steps in an ELISA sandwich method to detect circulating antigen that provides a rapid and reliable means of diagnosing the more severe forms of histoplasmosis (Wheat, 2003).

The role of humoral immunity in protection against H. capsulatum is uncertain. Early experiments indicated that passive immunization with immune serum did not mediate protection (Tewari et al., 1977). Also, B cell-deficient mice are not particularly susceptible to infection and high antibody titers do not correlate with resistance to infection (Allendorfer et al., 1999; Chandler et al., 1969). Consequently, the consensus in the field has traditionally been that humoral immunity has little or no role in host defense. Although murine mAbs to specific components of H. capsu-latum have been identified (Hamilton et al., 1990; Jeavons et al., 1994; Reiss et al., 1986), for most of them there are no published reports of studies evaluating their protective efficacy in mouse models of histoplasmosis (Nosanchuk, 2005). However, more recently Nosanchuk et al. described the generation of protective antibodies to H. capsulatum that bind to a histone H2B-like protein on the surface of the fungus (Nosanchuk et al., 2003). Administration of mAbs before infection reduced fungal burden, decreased pulmonary inflammation, and prolonged survival in a murine infection model. Protection was associated with enhanced levels of cytokines in the lungs of infected mice and the antibodies increased phagocytosis of yeast by macrophages through a CR3-dependent process leading to yeast cell growth inhibition and killing. The authors also suggested that that the histone H2B-like protein in H. capsulatum could be a potential candidate for vaccine development (Nosanchuk et al., 2003).

2.6. Blastomyces dermatitidis

Blastomycosis is an endemic mycoses in the central United States caused by a dimorphic fungus, Blastomyces dermatitidis. In nature this fungus exists in the mycelial phase but upon inhalation of spores into the lung, at body temperature, it converts to yeast phase. It has a wide spectrum of clinical manifestations, including asymptomatic self-limiting pulmonary illness, focal pulmonary or cutaneous illness, and disseminated disease (Bradsher et al., 2003).

Antibodies in human serum can mediate activation of complement that is required for killing of B. dermatitidis cells by human neutrophils (Ponton et al., 2001). Also, serological approaches can occasionally be helpful in establishing the diagnosis as antibodies to B. dermatiditis are produced in response to the infection. Early tests detected antibodies to Blastomyces A antigen, a yeast antigen (Klein et al., 1986), but recent efforts by Klein and others have focused on the BAD-1 antigen (previously designated WI-1), an important adhesin and virulence factor in B. dermatitidis, that is also the main antigenic target of humoral responses during human and experimental infections. (Klein and Jones, 1994; Rooney et al., 2001). Antibodies to this 120-kDa cell surface protein are detected earlier than A antigen and decline by 6 months after illness in patients who respond to therapy (Klein and Jones, 1994). Although vaccination with BAD-1 elicits strong antibody responses, protection was associated mostly with cell-mediated mechanisms (Wuthrich et al., 1998). Also, passive immunization with mAbs to BAD-1 did not protect animals against experimental infection (Wuthrich and Klein, 2000).

2.7. Coccidioides immitis and C. posadasii

Coccidioidomycosis is caused by the dimorphic fungi in the genus Coccidioides. These fungi live as mycelia in the soil of desert areas of the American Southwest. Humans acquire the infection by inhalation of the arthroconidia, which subsequently convert into the parasitic spherule/endospore phase. Most infections are mild, but these organisms are formidable pathogens capable of causing progressive pulmonary and/or disseminated disease in fully immunocompetent individuals (Cox and Magee, 2004).

Chronic or progressive coccidioidomycosis is associated with the generation of a strong polyclonal antibody response, with elevated levels of IgG, IgA, and IgE in serum of patients (Calhoun et al., 1986; Cox and Arnold, 1979; Cox et al., 1982; Pappagianis et al., 1965). Serum IgG levels directly correlate with disease involvement, being highest in patients with multifocal involvement, whereas serum IgA levels are elevated in approximately 20% of patients, mostly in those with chronic pulmonary disease (Pappagianis, 2001; Pappagianis and Zimmer, 1990). High titers of IgE consistent with a Th2 deleterious response have been demonstrated in approximately 23% of patients with active disease, with the highest incidence occurring in patients with disseminated disease. Serologic analyses using crude antigens prepared from filtrates or lysates of mycelial or spherule-endospore phases are useful in establishing a diagnosis of coccidioidomycosis and in determining prognosis (Pappagianis, 2001; Pappagianis and Zimmer, 1990). IgM antibodies may be detected within the first few weeks of infection, while IgGs are detected after a few weeks of infection and usually disappear in several months if the infection resolves (Pappagianis, 2001; Pappagianis and Zimmer, 1990). Serial antibody titers can be used to assess efficacy of therapy: rising titers are normally a poor prognostic sign whereas falling titers are associated with a disease resolution (Stevens, 1995).

Little is known about the potential role of antibody in the protection against coccidioidomycosis. Kong et al. (Kong et al., 1965) reported that passive transfer of serum from vaccinated mice (with formalin killed spherules, FKS) did not protect recipients. Beaman et al. (Beaman et al., 1977; Beaman et al., 1979) demonstrated that neither serum nor B cells from immune mice transferred protection in a murine model of coccidioidomycosis, and that preincubation of arthroconidia with serum from immune mice did not neutralize the infectivity of the arthrospores. Also, susceptible C57BL/6 mice produced higher titers of antibodies than resistant A/J did. Although several vaccine formulations containing Coccidioides killed cells (i.e. FKS), antigenic extracts or purified antigenic components (i.e. Ag2/PRA, SOW) have been tested in murine models, protection was generally associated with cell mediated immune mechanisms and not antibody responses (Cox and Magee, 2004). However, the possibility exists that there are protective antibodies that could be identified by using monoclonal antibody technology, as has been described for other fungal organisms.

2.8. Paraccoccidioides brasiliensis

P. brasiliensis is the etiologic agent of paracoccidioidomycosis, a prevalent systemic mycosis in South America where the lung is the primary target for infection. It is generally believed that antibodies are not effective in the control of chronic infection, but rather are abundant in anergic cases (de Camargo and de Franco, 2000).

The gp43 antigen, first described by Pucia et al. (Puccia et al., 1991), represents a major antigen of P. brasiliensis and forms the basis of a variety of serological tests, and antibody titers against gp43 can also be used to monitor therapeutic responses (Bueno et al., 1997). MAbs against gp43 modulate laminin-mediated fungal adhesion to epithelial cells and ameliorate pathogenicity in vivo either by inhibiting or enhancing granuloma formation and tissue destruction (Gesztesi et al., 1996). The gp70 is another immunodominant antigen in P. brasiliensis that can be used to monitor therapy in human infections (de Mattos Grosso et al., 2003). MAbs against gp70 were generated and exerted a protective effect against intratracheal infection, most likely through the inhibition of granuloma formation in the lungs (de Mattos Grosso et al., 2003). Also, de Fonseca et al. used immunoproteomics to identify six new antigens of P. brasiliensis that reacted with IgG antibodies present in sera from patients with paracoccidioidomycosis (da Fonseca et al., 2001).

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